| Four experiments were conducted to study survival of Bacillus subtilis natto in rumen and duodenum, dispear time and its effects on the performance, rumen fermentation, blood meterbolites and blood proteiniom.In experiment 1, experiment in vitro was conducted to evaluate the survival laws of Bacillus subtilis natto (BSN) in rumen and duodenum of Holstein dairy cows. BSN spores were added at 105/mL to strained rumen fluid or duodenum fluid taken from a healthy dairy cow and incubated in vitro at 39°C. Strained rumen fluid or duodenum fluid with no BSN inoculants served as controls. Changes of BSN spore counts and volatile fatty acid in rumen fluid were monitored at 0, 6, 12, 24, 48 and 72 h. Changes of BSN spore counts in duodenum fluid were monitored at 0, 1, 2, 3, 4, 5 and 6 h. The results of rumen fermentation showed that spores increased in the first 24 h, and then decreased. The spores at 24 h were two times of 0 h. In addition, BSN increased the concentration of propionate and butyrate in rumen fluid (P < 0.05), but reduced the concentration of acetic acid (P < 0.01). The results of duodenum fermentation showed that spore counts tended to increase in the first 2 h, and then decreased gradually.In experiment 2: seven cows were randomly assigned to two groups. Four cows were infused with 1010 spores of BSN into rumen through rumen cannula and the other three cows received no infusion. Rumen fluid, duodenum fluid and feces were collected at 0, 6, 12, 24, 48, 72 h after the infusion. Spore counts in rumen, duodenum and feces continued to decrease and almost can't be detected at 48 h after infusion in all of the location.In experiment 3: This experiment was conducted to determine the effect of B. subtillis natto fermentation product supplementation on blood metabolites, rumen fermentation, milk production and composition in early lactation dairy cows. Thirty-six multiparous Holstein cows (DIM = 29±6 d, parity = 2.8±1.1) were blocked by DIM and parity and then randomly assigned to 3 treatments (12 per treatment) in a 9-wk trial. Cows in control, DFM1 and DFM2 were fed TMR diets supplemented with 0, 6 or 12 gram of B. subtillis natto solid state fermentation product per day per cow respectively. Plasma nonesterified fatty acids were lower (P = 0.03) in DFM1 and DFM2 compared with control cows (633 and 639 vs. 685μmol/l). Ruminal propionate increased (P = 0.02) (23.9 vs. 26.3 and 26.9 mol/100 mol, control vs. DFM1 and DFM2, respectively) and acetate decreased (P < 0.05) (64.2 vs. 62.7 and 62.1 mol/100 mol, control vs. DFM1 and DFM2, respectively) with increasing B. subtillis natto fermentation product supplementation. DMI of the cows in three treatments was not affected by B. subtillis natto fermentation product supplementation, but milk yield was 3.1 kg/day and 3.2 kg/day higher for DFM1 and DFM2 than control cows on average across the 9-wk trial and significant differences were observed during wk 5 to 9 of the trial, which resulted in 9.5% and 11.7% increase in feed efficiency. B. subtillis natto fermentation product supplementation did not affect milk fat percentage and protein yield, but increased (P < 0.05) milk fat yield and lactose percentage (P < 0.01) and tended to decrease protein percentage (P = 0.06).In experiment 4: This experiment was conducted to study the effect of Bacillus subtilis natto on blood protein quantity and construction. Tweenty-four multiparous Holstein cows (DIM = 29±6 d, parity = 2.8±1.1) were blocked by DIM and parity and randomly assigned to 2 treatments (12 per treatment). Cows in control and DFM2 were fed TMR diets supplemented with 0 and 12 gram of B. subtillis natto solid state fermentation product per day per cow respectively. Changes of plasma proteins on the 8 wk after the fermentation product was fed were separated by two-dimensional electrophoresis providing a platform for parallel analysis. After proteins were stained with Coomassie Blue G-250, differentially expressed proteins were analyzed by PDQuest 7.4 software, and identified by HPLC equipped with ion trap mass spectrometer. The result showed that three proteins were up-regulated for the feeding of fermentation product; however, they were not identified. Transthyretin was down-regulated.In conclusion, B. subtillis natto spores have the ability to survive in rumen and alter rumen fermentation and survive in duodenum fluid for 3 h but can not permanently colonize in the gastrointestinal tract of Holstein dairy cows. The supplementation should be repeated in order to keep metabolic activities of B. subtillis natto additives and exert their effects on the ruminal microflora. When B. subtillis natto fermentation product was fed to dairy cows in early lactation, it was found that fermentation product was effective in increasing lactation performance of early lactation dairy cows possibly by altering the rumen fermentation pattern and down-regulat the express of transthyretin without any negative effects on blood metabolites.
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